The identification of specific types of cells present in a biological sample comprises a fundamentally important aspect of scientific, diagnostic and medical endeavour. The means by which such identification can be achieved often involves ascertaining the type of molecules expressed on the surface of cells. Such cell surface expression patterns can be determined using standard methods known to those skilled in the art, typically involving exposure of cells to antibodies that are specific for certain cell surface molecules. Antibodies used for this purpose can be conjugated either directly or indirectly with a fluorochrome that emits a signal upon excitation with light of a certain wavelength. In this way, the presence and quantification of particular cell populations in a biological sample can be determined.
Such techniques have found particular application in the field of immunology, where different populations of immune cells can be identified in a biological sample based upon their pattern of cell surface expression. For example, it is known that many lymphocytes express CD45 on their surface, and that a particular population of lymphocytes known as T cells can also express various other cell surface molecules including CD4 and CD25 depending upon their level of activation and development.
CD4+ T cells comprise a heterogeneous population of T cells which are of fundamental importance in both the generation of immune responses and the suppression of autoimmune diseases. A distinct subpopulation of CD4+ T cells also express CD25 and the transcription factor Foxp3. This subpopulation, loosely defined as regulatory T cells (Treg), plays a pivotal role in maintaining self tolerance (1). While the best evidence for the importance of Treg comes from mouse models, an increasing number of reports have outlined disturbances in Treg number and/or function in human patients with a wide variety of autoimmune (2-8), immunoinflammatory (9) and allergic diseases (10, 11), in addition to the very severe IPEX (immune dysregulation, polyendocrinopathy, enteropathy, and X-linked inheritance) syndrome in which the master regulator Foxp3 transcription factor itself is defective (12). Disturbances of Treg numbers have also been reported in cancers such as hepatocellular carcinoma (13) and head and neck cancer (14).
Hence, there is a clear need for a means of accurately identifying and quantifying Treg populations within in a biological sample. However, it has proven difficult to accurately distinguish Treg from CD25+ activated and memory T cells, particularly in human peripheral blood in which up to 20% of antigen-experienced CD4+ T cells also express CD25 (15). Such confusion in determining the precise identity of T cell populations has resulted in several studies reporting ambiguous results. For example, some studies have demonstrated an apparent reduction in Treg numbers in autoimmune conditions (2, 3, 8, 16), while others have shown normal or even increased numbers of CD4+CD25+ T cells (17-20).
The inventors have shown that a subpopulation of adult human naive CD4+CD25+ Treg cells derived from the thymus can be distinguished from the large population of CD25+ antigen-experienced conventional T cells on the basis of expression of CD45RA+/RO− (21). This naïve Treg population is reduced in young patients with inflammatory bowel disease (IBD), consistent with the existence of a primary deficiency in Treg production in these patients (22). However testing by the inventors with 38 different monoclonal antibodies failed to provide any means of separating human CD45RA−/RO+ Treg from activated/memory CD45RA−/RO+ T cells (21). Many of these monoclonal antibodies had previously been claimed to provide an accurate means of identification of CD45RA−/RO+ Treg (15, 23). It is therefore apparent that there is a need for improved methods of identifying Treg populations.
As both antigen-experienced CD4+ T cells and CD4+ Treg can also express CD25, conventional methods of identifying Treg on the basis of CD4/CD25 status are inadequate, leading to the potential for misinterpretation of data and incorrect associations of particular T cell populations with particular disease states. Indeed, at least part of the controversy surrounding the question of whether patients with allergic, autoimmune and immunoinflammatory diseases have primary deficiencies in Treg number or function stems from the difficulty in accurate identification of Treg. The range of constitutive CD25 expression by human Treg overlaps that of antigen-experienced activated/memory CD4+ cells. In addition, Foxp3, although crucial for the development of Treg, is also expressed by activated T cells, and thus fails to provide clear separation of Treg and activated/memory CD4+ cells (24). Hence, there is clearly a need for an improved method of accurately identifying and quantifying Treg populations.
The present invention is predicated on the surprising and unexpected finding by the inventors that Treg populations can be accurately defined by assessing the level of CD127 expressed on the surface of said populations.